Alteration of iron regulatory proteins (IRP1 and IRP2) and ferritin in the brains of scrapie-infected mice

Abstract

Considerable evidence suggests that oxidative stress may be involved in the pathogenesis of Transmissible Spongiform Encephalopathies (TSEs). To investigate the involvement of iron metabolism in TSEs, we examined the expression levels of iron regulatory proteins (IRPs), ferritins, and binding activities of IRPs to iron-responsive element (IRE) in scrapie-infected mice. We found that the IRPs-IRE-binding activities and ferritins were increased in the astrocytes of hippocampus and cerebral cortex in the brains of scrapie-infected mice. These results suggest that alteration of iron metabolism contributes to development of neurodegeneration and that some protective mechanisms against iron-induced oxidative damage may occur during the pathogenesis of TSEs.

Fig. 1

Western blots of protein expression levels of IRP1 (A) and IRP2 (B) in the brains of control (n = 18) and scrapie-infected mice (n = 18). The IRP1 and IRP2 protein expression levels in (A and B) were measured by densitometry (C); expression levels were increased by 2.0-fold and 2.1-fold, respectively, in the brains of scrapie-infected mice compared with control mice. *Values significantly different from control (p < 0.05). Expression of the housekeeping gene, β-actin, was measured in (A and B).

Fig. 2

Immunohistochemical localization of IRP1 and IRP2 in the brains of control and scrapie-infected mice. Intense immunolabeling of IRP1 and IRP2 appear in the hippocampus and cerebral cortex of scrapie-infected mice. Black arrows: vacuoles, white arrows: immunolabeled cells with IRP1 (B and E) and IRP2 (H and K). In (C, F, I and L) white arrows indicate immunolabeling of GFAP and black arrows indicate vacuoles. In sequential sections, immunoreactivity of IRP1 and IRP2 is colocalized with GFAP-positive reactive astrocytes. Sequential sections are B and C, E and F, H and I, K and L. Asterisk (*) indicates landmark blood vessels in the adjacent tissue section.

Fig. 3

IRE-binding activities of IRP1 and IRP2 in the brains of control (n = 12) and ME7 scrapie-infected C57BL mice (n = 12) by REMSA. IRE-binding activities of IRP1 and IRP2 are increased in scrapie-infected brains (lanes 4, 5, 8 and 9), compared with control brains (lanes 2, 3, 6 and 7). Lane 1: IRE probe only; lanes 2, 3, 6, 7 and 10: control; lanes 4, 5, 8, 9 and 11: scrapie-infected; lanes 6–9: full activation of IRPs detected by addition of 2% 2-ME; lanes 10 and 11: competition assay using 2-ME-treated brain samples as unlabeled IRE added to IRE probe: compare lanes 10 and 11 to 6, 7 and 8, 9, respectively. P: probe; C: control; I: infected; NS: non-specific bands.

Fig. 4

Protein expression levels of H/L- (A) and H-ferritin (B) in the brains of control (n = 18) and scrapie-infected mice (n = 18). The H/L- and H-ferritin protein expression in (A and B) were measured by densitometry in (C); expression levels were increased significantly in the brains of scrapie-infected mice compared with controls. *Values significantly different from control (*p < 0.05; **p < 0.01). Lanes 1–3: control; lanes 4–6: scrapie-infected mice. Expression of the housekeeping gene, β-actin, was measured in (A and B).

Fig. 5

Immunohistochemical localization of H/L- and H-ferritin in the hippocampus of control and scrapie-infected C57BL mice. Black arrows: vacoulation; white arrows: immunolabeled cells with H/L- (B) and H-ferritin (E). In (C and F) white arrows indicate GFAP immunolabeled cells and black arrows indicate vacuoles. In sequential sections, immunoreactivity for H/L- and H-ferritin is colocalized with GFAP-positive astrocytes. Sequential reactions are B and C, E and F. Asterisk (*) indicates landmark blood vessels in the adjacent tissue sections.